To activate inactive ARF-GDP, a guanine nucleotide-exchange protein (GEP) is required to promote GDP dissociation and GTP binding. This process can sometimes be inhibited by brefeldin A (BFA). BFA-sensitive and insensitive GEPs have been observed in both soluble and particulate cell fractions. Earlier, this group had purified two soluble BFA-insensitive GEPs and a BFA-inhibited GEP from bovine brain cytosol. Sequences of peptides from the BFA-sensitive GEP were used to clone a cDNA for the approx. 200-kDa protein. The deduced amino acid sequence contained a so-called Sec7 domain of 190 amino acids. Sec7 is an essential yeast gene involved in protein secretion and cytohesin-1, a protein abundant in cells of the immune system had been proposed to be a mammalian counterpart of yeast Sec7. It was shown rather quickly that cytohesin-1 has ARF GEP activity that is not inhibited by BFA. A His-tagged GEP synthesized in baculovirus-infected Sf9 cells using the BFA-sensitive GEP clone was inhibited by BFA, confirming that BFA sensitivity is an intrinsic property of this ARF GEP. The existence of two major families of ARF GEPs is now clear, the larger, approx. 200-kDa proteins that are inhibited by BFA and the approx. 50-kDa GEPs that are not. It was also shown that the His-tagged Sec7 domain from yeast Sec7 has BFA-inhibited GEP activity toward yeast ARF1 and ARF2, which function in protein secretion, but not yeast ARF3. Thus, the Sec7 domain contains also at least some of the determinants of ARF specificity. Arfaptin was identified and cloned in Exton's lab in a yeast two-hybrid screen with dominant negative ARF3 as bait and HL-60 cell cDNA library. They suggested that the 39-kDa arfaptin might be involved in Golgi function. Investigation of the effects of arfaptin on ARF activiation of cholera toxin and phospholipase D led to the conclusion that it acts as an inhibitor of the action of activated ARF, most effectively on myristoylated class I ARFs, but does not interfere with GEP activation of ARF-GDP.